Retroviral gene transfer, being highly efficient, is the most commonly used method for delivering therapeutic genes to hematopoietic cells. However, retrovirus vectors currently in use are not tissue-specific and therefore the target cells must be infected ex vivo , which necessitates removal of the target cells from the body maintenance and manipulation in culture, and subsequent re-transplantation. Furthermore, unless self- regenerating pluripotent stem cells are successfully transduced, the inserted genes are lost when the target cells undergo terminal differentiation; thus, extensive preliminary enrichment steps are required to achieve transduction of the rare hematopoietic stem cells. The aim of this proposal is to design strategies to overcome these problems and thereby improve the delivery of genes to hematopoietic cells by designing retroviral vectors which would specifically infect stem cells through ligand-receptor interaction. We previously created chimeric viral coat proteins consisting of the peptide hormone erythropoietin (EPO) inserted into the envelope gene of Moloney murine leukemia virus (MoMLV), and found that virus encoated by this targeting ligand showed a several-fold increase in infectivity for murine cells bearing the EPO receptor. More strikingly, with the EPOenv chimeric coat, this previously ecotropic (i.e, murine-specific) virus now became infectious for human cells bearing the EPO receptor. We plan to use the same principle to create retroviral vectors specific for hematopoietic stem cells, using as targets c-kit receptor and CD34. Three different targeting strategies will be employed: (1) a chimeric viral envelope consisting of stem cell factor (c-kit ligand) inserted into the envelope of ecotropic MoMLV, (2) the membrane-bound form of stem cell factor, which would be anchored to the cell membrane of virus-producing cells, with subsequent uptake and incorporation into the virion coat, and (3) a chimeric viral envelope which contains single chain antibody variable region sequences directed against cD34. These targeting ligands will be expressed in the ecotropic psi-2 packaging cell line, and the resultant virions will be assayed for specific infectivity on nonmurine target cells bering c-kit receptor and/or cD34, including human hematopoietic stem cells. Since the target cells are non-murine, any infection will be the result of a specific binding interaction between the targeting ligand on the viral surface and the c-kit receptor or CD34 on the target cell surface. The development of retroviral vectors which specifically target hematopoietic stem cells will have important implications for the treatment of sickle cell anemia and other genetic disorders. By achieving efficient gene transfer to pluripotent stem cells, these vectors will simplify preliminary preparation steps for in vivo infection, increase the likelihood of long term expression of the therapeutic gene, and may represent a delivery system which is potentially applicable in vivo.